PROJECT SUMMARY More than 1.8 million people are diagnosed with colorectal cancer (CRC) each year, and while increased rates of endoscopic screening have led to a slight reduction in mortality, the disease still accounts for more than 800,000 deaths worldwide annually. Treatments for CRC have changed little over the past 10-15 years, though immune checkpoint inhibitors (ICIs) have recently become an exciting therapeutic option for the treatment of hypermutated metastatic CRC (15% of all CRCs) and various other tumor types. However, for the remaining majority of CRC patients, there exist no effective targeted therapies. Clinical studies have revealed a correlation between WNT pathway activation and T-cell exclusion, that may explain why ICIs do not generate anti-tumor immune responses in CRC. Hyperactivation of the WNT signaling pathway is a hallmark and major oncogenic driver of CRC, occurring in ~95% of tumors. Early work from our lab in mice supports the notion that WNT signaling promotes immune suppression and regulates immune cell recruitment and that WNT pathway inhibition can block immune suppression in established tumors. Using precision models in the lab, I will test the hypothesis that WNT signaling drives immune suppression in CRC and inhibiting WNT signaling in CRC will activate tumor immune responses to ICIs. To address this question, I will utilize a model system of metastatic CRC using tumor organoid transplants?the disease setting in which ICIs are used in the clinic. Three organoid lines will be generated to have three distinct levels of WNT activation, all of which can regulated to undergo complete WNT pathway suppression. In Aim 1, I will determine the effect of distinct levels of WNT signaling on effector immune populations within colon tumors. Further, I aim to test whether WNT signaling suppression within established, WNT-active colon tumors facilitates recruitment of anti-tumor immune populations and reverses immune suppression. The genetic approach I am employing allows for inducible and reversible control of WNT-signaling modulation. This system is uniquely powerful to address my hypothesis as it allows an assessment of potent WNT activation and suppression within tumor cells. In Aim 2, I aim to determine if WNT signaling suppression stimulates immune response to ICIs resulting in tumor regression. I will first evaluate immune responses in tumors with tumor intrinsic WNT suppression. I will then assess pharmacological WNT pathway inhibition, that targets non-specifically but is clinically relevant, in its ability to recapitulate the genetic findings. Identifying a safe and effective approach to stimulate anti-tumor immunity will have a profound impact on the clinical management of CRC. Thus, we believe our work will contribute significant pre-clinical data to develop combination therapies for the activation of immunotherapies in CRC and potentially other tumor types with activated WNT signaling.